The Ras superfamily of GTPases comprises the Ras, Rho, Rab, Arf and Ran subfamilies (Colicelli, 2004 Sci. STKE. 2004:RE13.). Within the Ras subfamily, the RGK family includes four members, Rad (Reynet and Kahn, 1993 Science. 262:1441-4), Rem (Finlin and Andres, 1997 Mol. Biol. Cell. 9:1449-63), Rem2 (Béguin et al., 2005, Biochem. J. 390:67-75) and Kir/Gem (Cohen et al., 1994 Proc. Natl. Acad. Sci. U.S.A. 91:12448-52). RGK small GTP-binding proteins regulate cellular processes as diverse as voltage gated calcium channel (VGCC) activity (Béguin et al., 2006 J. Mol. Biol. 355:34-46) and actin and microtubule dynamics (Pelosi et al., 2007 Mol. Cell. Biol. 27:6163-76).
Kir/Gem, a member of the multifunctional RGK small GTP-binding protein family, regulates VGCC activity and actin and microtubule cytoskeleton remodeling. Individual functions of Kir/Gem are dependent on its subcellular distribution. Nuclear localization, for example, prevents its inhibitory effect on the Rho-Rho kinase pathway associated with changes in cell shape without affecting downregulation of VGCC activity. The subcellular distribution of Kir/Gem is modulated by calmodulin, 14-3-3 and importin binding, which in turn is regulated by phosphorylation.
Despite conserved core domains for nucleotide binding, members of the RGK family show striking structural and functional differences to other proteins within the Ras subfamily. While Ras switches between an inactive GDP-bound and an active GTP-bound conformation, unique structural features in the Ras core domain suggest an unconventional mechanism of inactivation for RGK proteins (Kelly, 2005 Trends Cell Biol. 15:640-3). Another distinctive feature of RGK proteins when compared to Ras is their regulation at the transcriptional level. Kir/Gem expression is induced upon transformation of cells with the abl tyrosine kinase oncogene (Cohen et al., 1994 Proc. Natl. Acad. Sci. U.S.A. 91:12448-52) and the stimulation of endothelial cells with IL-1α or TNFα (Warton et al., 2004 Gene. 342:85-95) or of peripheral T cells with mitogens (Maguire et al., 1994 Science. 265:241-4). Deregulation of Kir/Gem and Rad were also reported in neuroblastoma (Leone et al., 2001 Oncogene. 20:3217-25) and breast cancer (Tseng et al., 2001 Cancer Res. 61:2071-9) cell lines, respectively. The short half-life of the mRNA (˜30 min) (Cohen et al., 1994 Proc. Natl. Acad. Sci. U.S.A. 91:12448-52) and protein (˜3 hrs) (Ward et al., 2004 Mol. Cell. Biol. 24:651-61) suggests that RGK protein levels are tightly regulated in response to specific stimuli.
RGK proteins are multifunctional regulators of several cellular processes, including ion channel activity and cell shape remodeling. Binding of RGK proteins to the auxiliary β-subunit regulates the activity of VGCC, also known as voltage-dependent calcium channels or VDCC, either by interfering with cell surface transport of VGCCs or suppressing Ca2+ influx at the plasma membrane.
Voltage-gated calcium channels are a major route of calcium translocation across the plasma membrane of excitable cells. Intracellular calcium plays an important role in many biological processes such as calcium dependant neurotransmitter release, hormone secretion, muscle contraction and gene expression. More specifically, it is documented that abnormal levels of intracellular calcium create an imbalance in calcium homeostasis in a variety of cells, tissues and organs leading to many disorders. The conversions of the intracellular calcium flow by voltage-gated calcium channels is thought to impact a wide spectrum of biological responses and are implicated in several diseases, including ataxia, migraine, epilepsy, neurodegeneration, hypertension, cardiac disorders and diabetes. Currently there are several Calcium channel blockers used to treat some of these conditions. Compounds such as verapamil, isradipine, nefedipine, dilantizem and 1,4-dihydropine analogs of nefedipine interact with the L-type calcium channel to block calcium translocation and are widely used as antihypertensives, migraine treatment and in the treatment of certain vascular disorders. However, there are reports that therapeutic use of many calcium channel blockers is associated with potentially life-threatening side-effects. These include hypotension, constipation, decrease in insulin secretion leading to diabetes and heart block. Other calcium cannel blockers under development for use in disorders of the central nervous system and as analgesics include toxins that have been isolated from marine snails, scorpions, funnel web and tarantula spiders. The side effects and efficacy of such compounds are as yet unknown. Only two compounds have been found to act as agonists of voltage gated calcium channels, a dihydropyridine derivative BayK 8644 and Glycerotoxin isolated from sea worms. Despite the anticipated therapeutic effects of these compounds such as stimulating insulin secretion in diabetic and pre-diabetic living beings there has been severe side effects such as dystonic neurobehavioural syndrome, hypertension and arrhythmia during in vivo studies using BayK 8644 resulting in halted development of such compounds. There is a need for new agonists and antagonists capable of modulating voltage gated calcium channels to treat disorders associated with voltage gated calcium channels.
RGK proteins also act on the microtubule and actin cytoskeleton and their overexpression induces dendrite like extensions in COS-1 cells neurite outgrowth in neuroblastoma cells (Kir/Gem) and protrusions in endothelial cells (Rem). As shown for Kir/Gem and Rad, negative regulation of the Rho pathway by direct binding to Rho kinase and the Rho activating protein Gmip may account to a large extent for the effects of RGK proteins on cell morphology. Little is known about cell morthogenisis and the factors controlling cell shape and size.
Muscle formation involves the gradual differentiation of myocytes, which eventually fuse and exit the cell cycle to form multinucleated myotubes (Taylor, 2002 Curr. Biol. 12:R224-8). Myf5 and MyoD are primary muscle development factors (MRFs) required for the formation of skeletal myoblasts, whereas the secondary MRFs myogenin and MRF4 act as differentiation factors at later stages (Parker et al., 2003 Nat. Rev. Genet. 4:497-507). Myogenic differentiation relies on the activation of several signaling cascades, including the Rho-Rho kinase pathway (Bryan et al., 2005 Cell. Mol. Life. Sci. 62:1547-55) and apoptotic signals leading to the activation of caspases 3 and 12 (Fernando et al., 2002 Proc. Natl. Acad. Sci. U.S.A. 99:11025-30).
Many heart diseases result from a deficiency in the number of cardiomyocytes. Induction of new cardiomyocytes can possibly regenerate cardiac growth and potentially be used to treat such diseases. Heart tissue is the archetypical nonregenerative organ which is apparent from the rarity of cardiomycyte tumors. A number of approaches have been used to regenerate myocardium in experimental animals. Some of these include genetic manipulation of cardiomycytes, transplantation of skeletal myoblasts, use of donor cardiomycytes or the use of various stem cell transplants (Rubart M & Field L. J. 2006. annu rev. Physiol. 68:29-49). Of these methods the use of donor cardiomycytes has been most successful however there are several obstacles to the successful use of such cells in therapy. The cells do not increase in volume and may not survive transplantation further effecting the volume of these cells, it is very difficult to have sufficient numbers of donor cells and there is a lack of adequate immune suppression to efficiently minimise rejection of the cells. Further it is difficult to study differentiation of cydiomyocytes as they only represent 20% of the cells present in an adult ventrical.
RGK small GTP-binding proteins regulate cellular processes as diverse as VGCC activity (Yada et al., 2007 Circ. Res. 101:69-77) and actin and microtubule dynamics (Piddini et al., 2001 EMBO J. 20:4076-87). This results in a wide variation in subcellular distribution of RGK proteins which can translocate to the nucleus, with differential effects on RGK protein function. RGK proteins still downregulate channel activity when they localize to the nucleus, but no longer affect cell shape (Béguin et al., 2006 J. Mol. Biol. 355:34-46). This coupling between specific function and subcellular localization requires tight regulation, which is accomplished by posttranslational modifications and the association of regulatory factors. Nuclear localization is mediated by the association of importins with NLSs on RGK proteins. The relevance of nuclear localization is not well understood but the presence of endogenous nuclear Kir/Gem and Rem2 in primary hippocampal neurons as well as in situ in cells of the brain and heart indicate it is important.
The present invention seeks to provide novel modulators of RGK small GTP-binding proteins to vary concentration and location in a cell for use in treating disorders related to RGK small GTP-binding proteins concentration and location in a cell.
Those skilled in the art will appreciate that the invention described herein is susceptible to variations and modifications other than those specifically described. The invention includes all such variation and modifications. The invention also includes all of the steps, features, formulations and compounds referred to or indicated in the specification, individually or collectively and any and all combinations or any two or more of the steps or features.
Each document, reference, patent application or patent cited in this text is expressly incorporated herein in their entirety by reference, which means that it should be read and considered by the reader as part of this text. That the document, reference, patent application or patent cited in this text is not repeated in this text is merely for reasons of conciseness.
Any manufacturer's instructions, descriptions, product specifications, and product sheets for any products mentioned herein or in any document incorporated by reference herein, are hereby incorporated herein by reference, and may be employed in the practice of the invention.
The present invention is not to be limited in scope by any of the specific embodiments described herein. These embodiments are intended for the purpose of exemplification only. Functionally equivalent products, formulations and methods are clearly within the scope of the invention as described herein.
The invention described herein may include one or more range of values (eg size, concentration etc). A range of values will be understood to include all values within the range, including the values defining the range, and values adjacent to the range which lead to the same or substantially the same outcome as the values immediately adjacent to that value which defines the boundary to the range.
Throughout this specification, unless the context requires otherwise, the word “comprise” or variations such as “comprises” or “comprising”, will be understood to imply the inclusion of a stated integer or group of integers but not the exclusion of any other integer or group of integers. It is also noted that in this disclosure and particularly in the claims and/or paragraphs, terms such as “comprises”, “comprised”, “comprising” and the like can have the meaning attributed to it in U.S. patent law; e.g., they can mean “includes”, “included”, “including”, and the like; and that terms such as “consisting essentially of” and “consists essentially of” have the meaning ascribed to them in U.S. patent law, e.g., they allow for elements not explicitly recited, but exclude elements that are found in the prior art or that affect a basic or novel characteristic of the invention.
Other definitions for selected terms used herein may be found within the detailed description of the invention and apply throughout. Unless otherwise defined, all other scientific and technical terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which the invention belongs.